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. 2017 Jan 27;292(4):1240-1250.
doi: 10.1074/jbc.M116.748871. Epub 2016 Dec 9.

A Novel Rac1-GSPT1 Signaling Pathway Controls Astrogliosis Following Central Nervous System Injury

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Free PMC article

A Novel Rac1-GSPT1 Signaling Pathway Controls Astrogliosis Following Central Nervous System Injury

Taiji Ishii et al. J Biol Chem. .
Free PMC article

Abstract

Astrogliosis (i.e. glial scar), which is comprised primarily of proliferated astrocytes at the lesion site and migrated astrocytes from neighboring regions, is one of the key reactions in determining outcomes after CNS injury. In an effort to identify potential molecules/pathways that regulate astrogliosis, we sought to determine whether Rac/Rac-mediated signaling in astrocytes represents a novel candidate for therapeutic intervention following CNS injury. For these studies, we generated mice with Rac1 deletion under the control of the GFAP (glial fibrillary acidic protein) promoter (GFAP-Cre;Rac1flox/flox). GFAP-Cre;Rac1flox/flox (Rac1-KO) mice exhibited better recovery after spinal cord injury and exhibited reduced astrogliosis at the lesion site relative to control. Reduced astrogliosis was also observed in Rac1-KO mice following microbeam irradiation-induced injury. Moreover, knockdown (KD) or KO of Rac1 in astrocytes (LN229 cells, primary astrocytes, or primary astrocytes from Rac1-KO mice) led to delayed cell cycle progression and reduced cell migration. Rac1-KD or Rac1-KO astrocytes additionally had decreased levels of GSPT1 (G1 to S phase transition 1) expression and reduced responses of IL-1β and GSPT1 to LPS treatment, indicating that IL-1β and GSPT1 are downstream molecules of Rac1 associated with inflammatory condition. Furthermore, GSPT1-KD astrocytes had cell cycle delay, with no effect on cell migration. The cell cycle delay induced by Rac1-KD was rescued by overexpression of GSPT1. Based on these results, we propose that Rac1-GSPT1 represents a novel signaling axis in astrocytes that accelerates proliferation in response to inflammation, which is one important factor in the development of astrogliosis/glial scar following CNS injury.

Keywords: CNS injury; GSPT1; Rac (Rac GTPase); astrocyte; cell cycle; cell migration; cell proliferation; glial cell; inflammation; mouse.

Figures

FIGURE 1.
FIGURE 1.
Rac1-KO in astrocytes. A, RT-PCR was performed using cDNA obtained from WT primary astrocytes and specific primer-pairs of Rac1, Rac2, and Rac3. The predicted sizes of the amplified Rac1, Rac2, and Rac3 bands are 454, 581, and 440 bp. NC, negative control (without cDNA). B, primary astrocytes obtained from control and GFAP-Cre;Rac1flox/flox (Rac1-KO) mice were subjected to immunoblotting using a Rac1 antibody. Comparable loading of proteins was confirmed using tubulin-α antibody. C, spinal cords obtained from GFAP-Cre;Rac1flox/+;tdTomato mice were subjected to immunostaining using a GFAP antibody followed by Alexa 488 secondary antibody and then observed under a confocal laser microscope. Scale bar, 200 μm. D, magnified images of the area indicated by the rectangles in C are shown. Scale bar, 100 μm. E, spinal cords obtained from control (Rac1flox/+;tdTomato) mice were observed under a confocal laser microscope. DIC, differential interference image. Scale bar, 200 μm.
FIGURE 2.
FIGURE 2.
Better recovery of locomotor function after SCI and reduced astrogliosis after CNS injuries in Rac1-KO mice. A, BMS and BSS scores were recorded from days 1 to 35 after SCI. From day 9 after SCI, both hind limb movement and body support capability in Rac1-KO mice were significantly better than in control (cont) mice. This significant difference was sustained until day 35 (control; n = 14 hind limbs, Rac1-KO; n = 10 hind limbs; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Bonferroni's post hoc test following two-way ANOVA). B, sagittal sections of spinal cords from control and Rac1-KO mice 35 days after SCI were immunostained using a GFAP antibody and Alexa 488 secondary antibody. Immunoreactivity to GFAP in the area 100-μm rostral and caudal from the edge of the lesion (indicated by red lines) is shown (control; n = 12 sections from 3 mice, Rac1-KO; n = 12 sections from 4 mice; *, p = 0.0420). Scale bars, 100 μm. C, coronal sections of the right cerebellum and brainstem from control and Rac1-KO mice 21 days after microbeam irradiation injury (horizontally propagating multibeams, 100-μm width with 400-μm gaps between them) were immunostained using a GFAP antibody. GFAP-positive immunoreactivity in a linear band surrounding the irradiated lesions of the brainstem is shown (control; n = 9 ROIs from 3 mice, Rac1-KO; n = 9 ROIs from 3 mice; **, p = 0.0046). The right panels are magnified images of the regions indicated by the rectangles in the left panels. Scale bars, 500 μm.
FIGURE 3.
FIGURE 3.
Delayed cell cycle and impaired migration in Rac1-KD LN229 astrocytic cells. A, 48 h after transfection of LN229 cells (pSUPER (sh-cont) or shRac1(618)), efficacy of shRac1(618) on Rac1 expression levels was evaluated using a Rac1 antibody. Comparable loading of proteins was confirmed using a tubulin-α antibody. B, from 24 to 96 h after transfection (pSUPER(rfp) (sh-cont) or shRac1(618rfp)), the cell cycle of the RFP-expressing cells was evaluated using a Fucci system (upper left panel) and an LCV110 microscope. The cell cycle time (i.e. doubling time) is shown in the upper right panel (control: n = 57, Rac1-KD: n = 39; **, p = 0.0003). The lower left panel shows the cell cycle time of the G1 phase (control: n = 15, Rac1-KD: n = 16). The lower right panel shows the cell cycle time from the S to M phase (control: n = 15, Rac1-KD: n = 16; *, p = 0.0292). C, from 48 to 96 h after transfection (pSUPER(gfp) (sh-cont) or shRac1(618gfp)), migration capabilities of the GFP-expressing cells were monitored using an LCV110 microscope (0 h: starting time point, 48 h: ending time point, control: n = 17, Rac1-KD: n = 31; **, p < 0.0001). The white lines are located at the same position at time 0 and 48 h to show the movement (indicated by purple arrows at 48 h) of the cells marked by filled purple circles at 0 h. Scale bar, 200 μm.
FIGURE 4.
FIGURE 4.
Delayed cell cycle and impaired migration in both Rac1-KD and Rac1-KO primary astrocytes. A, left panel, 60 h after electroporation of 20 nm siRNA (si-cont or siRac1(618) + GFP plasmid) into WT primary astrocytes, Rac1 expression levels were evaluated using a Rac1 antibody. Comparable loading of proteins was confirmed using a GAPDH antibody. Right panel, primary astrocytes obtained from Rac1flox/flox;tdTomato (control, cont) and GFAP-Cre;Rac1flox/flox;tdTomato (Rac1-KO) mice were subjected to immunoblotting using a Rac1 antibody. Comparable loading of proteins was confirmed using a GAPDH antibody. B, the cell cycle was evaluated using an LCV110 microscope from 48 to 120 h after electroporation in the experiment using WT primary astrocytes (siRNA + GFP plasmid) or after the preparation on a glass-bottomed dish in the experiment using primary astrocytes from Rac1-KO (with tdTomato) and control mice. The left pair and the right pair in the graph show data obtained using Rac1-KD astrocytes (control: n = 79, Rac1-KD: n = 40; **, p = 0.0003) and using Rac1-KO astrocytes (control: n = 80, Rac1-KO: n = 82; **, p < 0.0001), respectively. C, 48–120 h after electroporation in the experiment using WT primary astrocytes (siRNA + GFP plasmid) or in the preparation on the glass-bottomed dish in the experiment using primary astrocytes from Rac1-KO (with tdTomato) and control mice, cell migration capabilities were evaluated using an LCV110 microscope. The left pair and the right pair in the graph show data obtained using Rac1-KD astrocytes (control: n = 31, Rac1-KD: n = 65; **, p < 0.0001) and using Rac1-KO astrocytes (control: n = 47, Rac1-KO: n = 39; **, p < 0.0001), respectively. D, 24 h after preparing the primary astrocytes from control and Rac1-KO mice in 24-well insets, cell migration capabilities were assayed using a CytoSelect migration assay kit (control: n = 10, Rac1-KO: n = 6; **, p < 0.0001).
FIGURE 5.
FIGURE 5.
Reduced expression of GSPT1 in Rac1-KD and -KO astrocytes. A, primary astrocytes obtained from control and Rac1-KO mice were treated with or without LPS (0.5 μg/ml) for 24 h. Expression levels of IL-1β were evaluated by immunoblotting using an IL-1β antibody. Rac1-KO and comparable loading of proteins were confirmed using a Rac1 antibody and tubulin-α antibody, respectively. The arrow indicates the IL-1β bands. B, LN229 astrocytic cells transfected with pSUPER (sh-cont) or shRac1(618) were treated with LPS (0.5 μg/ml) for 24 h. Reduced expression levels of Rac1 and IL-1β and comparable loading of proteins were confirmed using a Rac1 antibody, IL-1β antibody, and tubulin-α antibody, respectively. The arrow indicates the IL-1β bands. C, Rac1 was knocked down via transfection of 2.5 nm of siRNAs (si-cont, siRac1(618), or siRac1(1977)) in LN229 cells. Primary astrocytes were prepared from control and Rac1-KO mice. Reduced expression levels of GSPT1 were evaluated using a GSPT1 antibody. Rac1-KD/KO and comparable loading of proteins were confirmed using a Rac1 antibody and GAPDH antibody, respectively. D, 2.5 nm of siRNAs (si-cont or siRac1(618)) were transfected in LN229 cells 24 h prior to LPS treatment. After LPS treatment (0.5 μg/ml) for 24 h, GSPT1 levels were evaluated using a GSPT1 antibody. Rac1-KD and comparable loading of proteins were confirmed using a Rac1 antibody and GAPDH antibody, respectively. E, LN229 cells were simultaneously treated with LPS (1.0 μg/ml) and one of four inhibitors at the indicated concentrations (μm; JNK-IN-8, SB203580, U0126, or BAY 11-7085) for 16 h. After the treatment, GSPT1 levels were evaluated using a GSPT1 antibody. Comparable loading of proteins was confirmed using a GAPDH antibody.
FIGURE 6.
FIGURE 6.
Cell cycle delay by GSPT1-KD and rescue of cell cycle delay induced by Rac1-KD via overexpression of GSPT1. A, 5 and 10 nm of control (si-cont) or two GSPT1 siRNAs (si620 or si1374) were co-transfected with Venus-hGeminin plasmid into HeLa cells. At 48 h after transfection, expression levels of GSPT1 were evaluated using a GSPT1 antibody. Comparable loading of proteins was confirmed using a GAPDH antibody. B, 14–96 h after transfection (10 nm of siRNA + Venus-hGeminin plasmid) into HeLa cells, the cell cycle time of Venus-hGeminin transfected cells was observed under an LCV110 microscope (si-cont: n = 68, si620: n = 85, si1374: n = 56; **, p < 0.0001). C, 2.5 and 5 nm of si-cont or siRac1(618) was co-transfected with the GFP plasmid into HeLa cells. For the rescue experiment, Rac1 siRNA + GFP-GSPT1 plasmid was co-transfected into HeLa cells. At 48 h after transfection, expression levels of Rac1, GSPT1, overexpressed GFP-GSPT1, and GFP were examined by immunoblotting using a Rac1, GSPT, and GFP antibody, respectively. Comparable loading of proteins was confirmed using a GAPDH antibody. D, from 24 to 96 h after transfection (siRac1(618) + GFP or GFP-GSPT1 plasmid) into HeLa cells, the cell cycle time of GFP or GFP-GSPT1 transfected cells was observed under an LCV110 microscope (2.5 nm siRac1, GFP: n = 171, GFP-GSPT1: n = 171; **, p = 0.0003; and 5 nm siRac1, GFP: n = 148, GFP-GSPT1: n = 77; **, p < 0.0001). E, 10 and 20 nm of si-cont or siGSPT1(620m) were co-electroporated with the GFP plasmid into the primary astrocytes. 60 h after electroporation, the expression levels of GSPT1 were evaluated using a GSPT1 antibody. Comparable loading of proteins was confirmed using a GAPDH antibody. F, from 48 to 120 h after electroporation (20 nm of siRNA + GFP plasmid) of WT primary astrocytes, the cell cycle time of the GFP-transfected cells was assessed using an LCV110 microscope (si-cont: n = 107, si620m: n = 90; **, p = 0.0001).
FIGURE 7.
FIGURE 7.
No effect of GSPT1-KD on astrocyte migration. A, 5 and 10 nm of control (si-cont) or two GSPT1 siRNAs (si620 or si1374) were co-transfected with GFP plasmid into LN229 astrocytic cells. At 48 h after transfection, expression levels of GSPT1 were evaluated using a GSPT1 antibody. Comparable loading of proteins was confirmed using a GAPDH antibody. B, from 48 to 96 h after transfection (10 nm of siRNA + GFP), the cell migration capabilities of the LN229 cells were monitored using an LCV110 microscope (si-cont: n = 51, si620: n = 50, si1374: n = 50). C, 20 nm of si-cont or siGSPT1(620m) was co-electroporated with the GFP plasmid into WT primary astrocytes. 32 h after electroporation, the expression levels of GSPT1 were evaluated using a GSPT1 antibody. Comparable loading of proteins was confirmed using a GAPDH antibody. D, 32 h after electroporation (20 nm of siRNA + GFP plasmid) into WT primary astrocytes, cell migration capabilities were assayed using a CytoSelect migration assay kit (si-cont: n = 8, si620m: n = 4).

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